Proline ester and preparation containing the same for percutaneous administration

ABSTRACT

A proline ester represented by the following formula (I): 
                         
wherein R 1  represents a hydroxy-lower alkyl group, a lower alkoxy-lower alkyl group, or a lower alkoxy-lower alkoxy-lower alkyl group or a pharmaceutically acceptable salt thereof.
 
     The proline ester (I) of the present invention and a pharmaceutically acceptable salt thereof are useful as a prodrug of enalaprilat, which is a medicine useful for preventing or treating circulatory diseases such as hypertension, cardiac diseases (e.g., cardiac hypertrophy, cardiac failure, and myocardial infarct), nephritis, and apoplexy. Thus, a drug containing the ester or a salt thereof is preferably formulated to a percutaneous preparation, particularly a patch, from the viewpoint of medicinal activity and use.

This application is a 371 of PCT/JP03/11420 filed on Sep. 8, 2003.

TECHNICAL FIELD

The present invention relates to a proline ester which is useful as aprodrug of enalaprilat, an agent useful in treatment of hypertension orcardiac failure, to drugs containing the ester, and to preparationscontaining the ester for percutaneous administration.

BACKGROUND ART

Currently, as agents for the treatment of hypertension, a variety ofperoral preparations such as a calcium antagonist, anangiotensin-converting enzyme (ACE) inhibitor, and an angiotensinreceptor antagonist are employed in clinical settings. However, not asmall number of patients with hypertension tend to experience difficultyin swallowing or have disorders in the digestive tract, and in suchcases, peroral administration encounters difficulty. Therefore,development of an antihypertensive agent that can be administeredthrough a route other than peroral route has been awaited. In general,use of a percutaneous preparation is suitable means for treatingpatients having difficulty in peroral administration, since a drug isabsorbed through the skin. However, an antihypertensive agent in theform of percutaneous preparation has not become clinically available dueto poor percutaneous absorbability, etc.

Meanwhile, enalapril in the form of a peroral agent is widely used inclinical settings as an antihypertensive agent having an ACE inhibitoryeffect. Enalapril is a prodrug of enalaprilat, which is an activemetabolite of enalapril. Enalapril is formed throughethyl-esterification of one of the two carboxyl groups which is presentin the vicinity of the center of the enalaprilat molecule. Whenperorally administered, enalapril is absorbed via the digestive tractand metabolized in the liver, where it is converted to enalaprilat,which is useful as an ACE inhibitor exhibiting a therapeutic effect.

Enalapril and enalaprilat are percutaneously absorbable. However, thesecompounds serving as active ingredients contained in a percutaneouspreparation exhibit very poor physicochemical stability and therefore,they have not been successfully formed into percutaneous preparations.It has been proven that the poor stabilities of enalapril andenalaprilat are attributable to intramolecular ring formation throughcondensation of a secondary amine moiety of alanine with a carboxylgroup linked to the proline ring (Analytical Profiles of DrugSubstances, USA Academic press, Inc., 1987, Vol. 16, p. 207-244, DrugDevelopment And Industrial Pharmacy, USA, Marcel Dekker, Inc., 1986,Vol. 12, 14, p. 2467-2480). Furthermore, there has been known thatpercutaneously absorbed enalapril is not converted to enalaprilat in theskin and also in the plasma (Drug Metabolism And Disposition, USA, TheAmerican Society for Pharmacology and Experimental Therapeutics, 1982,Vol. 10, 1, p. 15-19). Therefore, active enalaprilat is formed only whenpercutaneously absorbed enalapril is circulated to the liver andmetabolized, which means that among other problems, a long period oftime is required to express ACE inhibitory effect, and consistenttherapeutic effect often fails to attain.

Research efforts have also been devoted to prodrug design; i.e.,chemical modification of enalaprilat so as to change to a drug havingphysical properties suitable for percutaneous absorption whilemaintaining the intrinsic drug effect. In one report, a prodrug isformed through ethyl-esterification of both the carboxyl group presentin the center of the enalapril molecule and the carboxyl group linked tothe proline ring. The prodrug is known to exhibit enhanced percutaneousabsorbability as compared with enalapril maleate (THE STUDY OFTRANSDERMAL ADMINISTRATION OF ACE INHIBITORS AND IMPROVED ABSORPTION OFTHEIR PRODRUGS,” [online], Nov. 2 (2000), Noven pharmaceuticals, Inc.,AAPS Annual Meeting and Exposition (2000),internet<URL:http://www.noven.com/Noven Doc3.pdf>). However, althoughthe ethyl ester moiety of the carboxyl group linked to the proline ringof the prodrug compound is hydrolyzed by an esterase present in thehuman skin at a hydrolyzation degree of 50% or more, the other ethylester moiety does not undergo hydrolysis. Therefore, similar toenalapril, the prodrug compound must be metabolized in the liver to forman active species, which is problematic.

DISCLOSURE OF THE INVENTION

In view of the foregoing, an object of the present invention is toprovide a prodrug of enalaprilat which exhibits excellent percutaneousabsorbability and physicochemical stability and which is readilyconverted to enalaprilat during the course of percutaneous absorption,so as to use enalaprilat via percutaneous absorption in clinicaltreatments. Another object of the invention is to provide a drugcontaining the prodrug. Still another object of the invention is toprovide a percutaneous preparation containing the prodrug.

In an attempt to attain the above objects, the present inventors havesynthesized and investigated a large number of candidate compoundsserving as a prodrug of enalaprilat, and have found that a compound inwhich a carboxyl group on the proline ring of enalaprilat is esterifiedby a lower alkyl group having a specific substituent exhibits excellentphysicochemical stability and skin-permeability and is effectivelyconverted to enalaprilat during the course of skin permeation, therebybeing useful for the prodrug, although a compound in which a carboxylgroup on the proline ring of enalaprilat is esterified by an alkyl groupexhibits no improvement in percutaneous absorbability. The presentinvention has been accomplished on the basis of this finding.

Accordingly, the present invention provides a proline ester representedby the following formula (I):

wherein R¹ represents a hydroxy-lower alkyl group, a lower alkoxy-loweralkyl group, or a lower alkoxy-lower alkoxy-lower alkyl group or apharmaceutically acceptable salt thereof. The invention also provides adrug containing the ester or the salt thereof, particularly apercutaneous preparation.

The present invention also provides use, for producing a drug, of aproline ester represented by the above formula (I) or a pharmaceuticallyacceptable salt thereof.

The present invention further provides a method for treating apathological condition affected or induced by activation of ACE,characterized by administering, to a patient in need thereof, a prolineester represented by the above formula (I) or a pharmaceuticallyacceptable salt thereof.

The proline ester (I) of the present invention and a pharmaceuticallyacceptable salt thereof are useful as a prodrug of enalaprilat, which isa drug useful for preventing or treating circulatory pathologicalconditions such as hypertension, cardiac diseases (e.g., cardiachypertrophy, cardiac failure, and myocardial infarct), nephritis, andapoplexy. Thus, a drug containing the ester or a salt thereof ispreferably formulated to a percutaneous preparation, particularly apatch, from the viewpoint of medicinal activity and use.

BEST MODES FOR CARRYING OUT THE INVENTION

Among substituents represented by R1 in formula (I) and R2 in the belowmentioned formula (II), the term “lower alkyl” refers to a C1 to C6linear or branched alkyl group, and the term “lower alkoxy” refers to aC1 to C4 linear or branched alkoxy group.

Examples of the hydroxy lower alkyl group represented by R1 includehydroxy-C1-6 alkyl groups such as hydroxymethyl, hydroxyethyl,hydroxypropyl, hydroxybutyl, hydroxypentyl, and hydroxyhexyl. Of these,2-hydroxyethyl, 3-hydroxypropyl, and 4-hydroxybutyl are preferred.

Examples of the lower alkoxy-lower alkyl group represented by R1 includeC1-4 alkoxy-C1-6 alkyl groups such as methoxyethyl, ethoxyethyl,propoxyethyl, isopropoxyethyl, butoxyethyl, isobutoxyethyl,sec-butoxyethyl, tert-butoxyethyl, and methoxypropyl. Of these,methoxyethyl is preferred.

Examples of the lower alkoxy-lower alkoxy-lower alkyl group representedby R1 include C1-4 alkoxy-C1-4 alkoxy-C1-6 alkyl groups such asmethoxymethoxyethyl, methoxyethoxyethyl, and methoxymethoxybutyl. Ofthese, methoxyethoxyethyl is preferred.

The compound (I) of the present invention includes a hydrate thereof,solvates thereof, and all crystal forms thereof.

Examples of the pharmaceutically acceptable salt of the compound (I) ofthe present invention include inorganic acid salts such ashydrochlorides, hydrobromides, hydroiodides, nitrates, sulfates, andphosphates; organic acid salts such as acetates, propionates,trifluoroacetates, oxalates, fumarates, maleates, tartrates, citrates,succinates, malates, methanesulfonates, benzenesulfonates, andp-toluenesulfonates; alkali metal salts such as lithium salts, sodiumsalts, and potassium salts; and alkaline earth metal salts such ascalcium salts and magnesium salts.

The compound (I) of the present invention is preferably1-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline 2-hydroxyethylester, 1-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline3-hydroxypropyl ester,1-[(N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline 4-hydroxybutylester, 1-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline2-(2-methoxyethoxy)ethyl ester, or1-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline 2-methoxyethylester.

The compound (I) of the present invention is produced through, forexample, the following reaction scheme:

wherein R² represents a benzyloxy-lower alkyl group, a loweralkoxy-lower alkyl group, or a lower alkoxy-lower alkoxy-lower alkylgroup, Bn represents a benzyl group, and R¹ has the same meaning asdescribed above.

Specifically, the compound (I) of the present invention may be producedthrough deprotection of a compound (II) by removing a benzyl group withcatalytic reduction (step A).

The reaction may be performed by subjecting the compound (II) tocatalytic reduction in a solvent such as methanol, ethanol, ether,tetrahydrofuran, dioxane, N,N-dimethylformamide, or a solvent mixturethereof, in the presence of a metal catalyst such as palladium carbon,palladium black, tris(triphenylphosphine)rhodium chloride, or platinumoxide, at a temperature between 0° C. and the boiling point of thesolvent, under hydrogen at normal or middle pressure. Through thisreaction, a benzyl ester moiety or a benzyl ether moiety can bedeprotected.

The compound (II), an intermediate compound for producing the compound(I) of the present invention, may be produced, through the followingreaction scheme, by tert-butyl-esterifying enalapril (III) therebypreparing a compound (IV) (step B); selectively hydrolyzing the ethylester moiety of the compound (IV) thereby preparing compound (V) (stepC); benzyl-esterifying the compound (V) thereby preparing compound (VI)(step D); hydrolyzing the tert-butyl ester moiety of the compound (VI)(step E), and reacting the resultant compound with R²OH (VIII):

wherein R² and Bn have the same meanings as described above.

In Step B, enalapril (III) is reacted with isobutene in a solvent suchas ether, tetrahydrofuran, dioxane, dichloromethane, chloroform, or asolvent mixture thereof in the presence of an acid catalyst such asconcentrated sulfuric acid or boron trifluoride diethyl etherate at atemperature between −78° C. and room temperature. In Step C, the estermoiety of the compound (IV) is hydrolyzed by use of an alkaline aqueoussolution such as an aqueous sodium hydroxide solution or an aqueouspotassium hydroxide solution. In Step D, the compound (V) is reactedwith a benzyl halide in a solvent such as ether, tetrahydrofuran,dioxane, N,N-dimethylformamide, dimethylsulfoxide, dichloromethane,chloroform, or a solvent mixture thereof in the presence of a base suchas sodium amide, lithium amide, sodium hydride, potassium carbonate, orpotassium tert-butoxide at a temperature between 0° C. and the boilingpoint of the solvent. In Step E, the tert-butyl ester moiety of thecompound (VI) is deprotected in a solvent such as ether,tetrahydrofuran, dioxane, dichloromethane, chloroform, or a solventmixture thereof in the presence of an acid such as formic acid, aceticacid, trifluoroacetic acid, hydrochloric acid, or a hydrogenchloride-dioxane solution at a temperature between 0° C. and the boilingpoint of the solvent. In Step F, the compound (VII) is reacted with analcohol (VIII) in a solvent such as benzene, toluene, xylene, or asolvent mixture thereof in the presence of an acid catalyst such asp-toluenesulfonic acid at the boiling point of the solvent.

Alternatively, the compound (II) may be prepared, through the followingreaction scheme, by hydrolyzing a compound (IX) thereby preparing acompound (X) (step G); benzyl-esterifying the compound (X) therebypreparing a compound (XI) (step H); reacting the compound (XI) with anL-alanine tert-butyl ester thereby preparing a compound (XII) (step I),selectively hydrolyzing the tert-butyl ester moiety of the compound(XII) (step J), and subjecting the compound (XIII) to condensation withthe compound (XIV) (step K):

wherein R² and Bn have the same meanings as described above.

In the step G, the ester moiety of a compound (IX) is hydrolyzed by useof an alkaline aqueous solution such as an aqueous sodium hydroxidesolution or an aqueous potassium hydroxide solution. In Step H, thecompound (X) is reacted with a benzyl halide in a solvent such as ether,tetrahydrofuran, dioxane, N,N-dimethylformamide, dimethylsulfoxide,dichloromethane, chloroform, or a solvent mixture thereof in thepresence of a base such as sodium amide, lithium amide, sodium hydride,potassium carbonate, potassium hydrogencarbonate, potassiumtert-butoxide at a temperature between 0° C. and the boiling point ofthe solvent. In Step I, the compound (XI) is reacted withtrifluoromethanesulfonic anhydride in a solvent such as dichloromethaneor chloroform in the presence of 2,6-lutidine to thereby prepare atrifluoromethanesulfonic acid ester, and the ester is reacted with anL-alanine tert-butyl ester hydrochloride. In Step J, the tert-butylester moiety of the compound (XII) is deprotected in a solvent such asether, tetrahydrofuran, dioxane, dichloromethane, chloroform, or asolvent mixture thereof in the presence of an acid such as formic acid,acetic acid, trifluoroacetic acid, hydrochloric acid, or a hydrogenchloride-dioxane solution at a temperature between 0° C. and the boilingpoint of the solvent. In Step K, the compound (XIII) is condensed with acompound (XIV) in a solvent such as ether, tetrahydrofuran, dioxane,N,N-dimethylformamide, dichloromethane, chloroform, benzene, toluene, ora solvent mixture thereof at a temperature between 0° C. and the boilingpoint of the solvent (if necessary, in the presence of a base such astriethylamine, ethyldiisopropylamine, or1,8-diazabicyclo[5.4.0]undec-7-ene) by use of a condensing agent such as1-hydroxy-1H-benzotriazole, N-hydroxysuccinimide,N,N′-dicyclohexylcarbodiimide, or1-ethyl-3-(3-dimethylaminopropylcarbodiimide).

The compound (XIV) may be prepared by reacting proline (XV) with analcohol (VIII) in a solvent such as benzene, toluene, xylene, or asolvent mixture thereof in the presence of an acid catalyst such asp-toluenesulfonic acid at the boiling point of the solvent (step L):

wherein R² has the same meaning as described above.

The thus-obtained compound (I) of the present invention may be isolatedor purified through a usual process such as recrystallization or columnchromatography.

The compound (I) of the present invention may be formed into a salt witha pharmacologically acceptable acid or base through a usual method.

The compound (I) of the present invention or a pharmaceuticallyacceptable salt thereof is converted to enalaprilat through percutaneousadministration and, therefore, exhibits excellent ACE-inhibitoryactivity. By virtue of the activity, the compound (I) or apharmaceutically acceptable salt thereof can be used for preventing ortreating pathological conditions affected or induced by activation ofACE, for example, circulatory diseases such as hypertension, cardiacdiseases (e.g., cardiac hypertrophy, cardiac failure, and myocardialinfarct), nephritis, and apoplexy. Although the daily dose of compound(I) administered per adult may vary depending on the pathologicalconditions, body weight, and age of patients, and type of compound,etc., the dose is preferably about 1 to about 1,000 mg.

Preferably, the percutaneous preparation of the present inventionfurther contains a percutaneous absorption enhancer for enhancingpercutaneous absorbability. The percutaneous absorption enhancer may beselected from the group consisting of fatty acid esters and nonionicsurfactants. These enhancers may be used singly or in combination of twoor more species.

Examples of the fatty acid ester include fatty acid esters formed from aC6 to C22 fatty acid and a C1 to C12 alcohol. Examples of the C6 to C22fatty acid include monocarboxylic acids such as caproic acid, enanthicacid, caprylic acid, capric acid, undecylenic acid, lauric acid,myristic acid, palmitic acid, margaric acid, stearic acid, oleic acid,and linoleic acid and dicarboxylic acids such as adipic acid and sebacicacid. Examples of the C1 to C12 alcohol include methanol, ethanol,propanol, isopropanol, butanol, tert-butanol, hexanol, octanol, and1-octyl dodecanol. Thus, examples of the fatty acid esters includediisopropyl adipate, diethyl sebacate, isopropyl myristate, isopropylpalmitate, isopropyl stearate, butyl stearate, octyldodecyl myristate,butyl myristate, hexyl laurate, octyl palmitate, and ethyl oleate. Ofthese, isopropyl myristate, isopropyl palmitate, diethyl sebacate, andthe like are preferred, with isopropyl myristate being particularlypreferred.

Examples of the nonionic surfactant include polyoxyethylene alkylethers, polyoxyethylene alkyl aryl ethers, fatty acid amides, glycerinfatty acid esters, propylene glycol fatty acid esters, sorbitan fattyacid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylenefatty acid esters, polyoxyethylene glycerin fatty acid esters,polyoxyethylene castor oil derivatives, block polymer type nonionicsurfactants, and polyglycerin fatty acid esters. Of these,polyoxyethylene alkyl ethers, fatty acid amides, glycerin fatty acidesters, sorbitan fatty acid esters, and polyoxyethylene sorbitan fattyacid esters are preferred.

Examples of the polyoxyethylene alkyl ethers include polyoxyethylenecetyl ether, polyoxyethylene oleyl ether, and polyoxyethylene laurylether. Particularly, polyoxyethylene lauryl ether (lauromacrogol) ispreferred.

Examples of the fatty acid amides include lauric acid monoethanolamide,lauric acid diethanolamide, and oleic acid diethanolamide. Particularly,lauric acid diethanolamide is preferred.

Examples of the glycerin fatty acid esters include glycerylmonocaprylate, glyceryl monolaurate, glyceryl monopalmitate, glycerylmonooleate, and glyceryl monostearate. Particularly, glycerylmonocaprylate and glyceryl monolaurate are preferred.

Examples of the sorbitan fatty acid esters include sorbitan monocaprylicacid ester, sorbitan monolauric acid ester, sorbitan monopalmitic acidester, sorbitan monooleic acid ester, and sorbitan monostearic acidester. Particularly, sorbitan monocaprylic acid ester is preferred.

Examples of the polyoxyethylene sorbitan fatty acid esters includepolyoxyethylene sorbitan monooleic acid ester, polyoxyethylene sorbitantrioleic acid ester, and polyoxyethylene sorbitan monopalmitic acidester. Particularly, polyoxyethylene sorbitan monooleic acid ester ispreferred.

The percutaneous absorption enhancer is preferably selected from thegroup consisting of isopropyl myristate, lauromacrogol, lauric aciddiethanolamide, glyceryl monocaprylate, glyceryl monolaurate, sorbitanmonocaprylic acid ester, and polyoxyethylene sorbitan monooleic acidester.

No particular limitation is imposed on the preparation form of thepercutaneous preparation, and examples of the form include ointment,cream, patch, and lotion. Of these, patch is preferred from theviewpoint of use of patients.

The type of the patch may include a known support and a known adhesivelayer, and may be produced by applying, to a surface of a support, anadhesive containing the compound of the present invention (I) or a saltthereof and an additive such as a percutaneous absorption enhancer, andcutting the coated support to pieces having predetermined sizes. Thesurface of the adhesive layer which is not in contact with the supportmay be coated with a protective member such as a peelable sheet.Alternatively, the surface may be protected by rolling the coatedsupport.

Examples of the adhesive employed in the invention include an acrylicadhesive, a rubber adhesive, and a silicone adhesive, which exhibitpressure-sensitivity at ambient temperature.

The acrylic adhesive preferably contains a homopolymer, for example,comprised of a (meth)acrylic acid alkyl ester as a predominantcomponent, or copolymer thereof with another comonomer (as used herein,the term “(meth)acrylic acid” refers to methacrylic acid or acrylicacid). Examples of the (meth)acrylic acid alkyl ester monomer include(meth)acrylic acid-2-ethylhexyl ester, (meth)acrylic acid ethyl ester,(meth)acrylic acid butyl ester, (meth)acrylic acid isobutyl ester,(meth)acrylic acid hexyl ester, (meth)acrylic acid octyl ester,(meth)acrylic acid decyl ester, (meth)acrylic acid isodecyl ester,(meth)acrylic acid lauryl ester, and (meth)acrylic acid stearyl ester.Examples of the comonomer include acrylic acid, methacrylic acid, maleicacid, fumaric acid, 2-hydroxyethyl(meth)acrylate, hydroxypropyl(meth)acrylate, acrylamide, dimethyl acrylamide, diethyl acrylamide,butoxymethyl acrylamide, ethoxymethyl acrylamide, N-vinyl-2-pyrrolidone,vinyl acetate, vinyl propionate, styrene, α-methylstyrene, vinylchloride, acrylonitrile, ethylene, propylene, and butadiene.

No particular limitation is imposed on the type of the rubber adhesive,and examples include styrene-butadiene copolymer, styrene-isoprenecopolymer, styrene-isoprene-styrene block copolymer, natural rubber,synthetic isoprene rubber, polyisobutylene, polyvinyl ether,polyurethane, polyisoprene, and polybutadiene.

No particular limitation is imposed on the silicone adhesive, andexample include silicone rubber such as polyorganosiloxane.

Among them, styrene-isoprene-styrene block copolymer is particularlypreferred, since the block polymer exhibits excellent compatibility withthe compound (I) of the present invention and excellent physicalproperties for serving as a patch.

The support is preferably resistive to drug permeation. Examples of thesupport include resin films made of polyethylene terephthalate,cellulose acetate, ethyl cellulose, nylon, ethylene-vinyl acetatecopolymer, polyethylene, or polyurethane. Since the support is requiredto have flexibility, the thickness of the support is generally 300 μm orless, preferably 2 to 100 μm or less.

The peelable sheet is required to be readily separated from the adhesivelayer upon use. Thus, the peelable sheet is generally formed of a filmcoated with silicone on the side which is in contact with the adhesivelayer. Examples of the film material include polyethylene terephthalate,polyvinyl chloride, polyvinylidene chloride, and polyester. A laminatefilm such as glassine paper sheet may also be used. The peelable sheetgenerally has a thickness of 1,000 μm or less, preferably 30 to 150 μm.

The patch contains the compound (I) of the present invention in anamount required for treatment. The compound (I) of the present inventionor a salt thereof is preferably incorporated into the adhesive layer inan amount of 0.1 to 30 mass % with respect to the total mass of theadhesive layer in a dried state, more preferably 0.5 to 20 mass %. Theadhesive layer preferably has a thickness of 10 to 400 μm.

The percutaneous absorption enhancer is preferably incorporated into theadhesive layer in an amount of 0.1 to 60 mass % with respect to thetotal mass of the adhesive layer in a dried state, more preferably 1 to40 mass %. In the case of a fatty acid ester, the amount is preferably 1to 40 mass %, and in the case of a non-ionic surfactant, the amount ispreferably 1 to 20 mass %.

No particular limitation is imposed on the method for forming theadhesive layer of the patch, but a solution coating method is preferred.Specifically, an adhesive, a drug, an optional percutaneous absorptionenhancer, and optional additives are mixed together, and the mixture isdispersed in an organic solvent. The obtained dispersion is applied to asurface of the support by use of an applicator, and the coating is driedto remove the solvent, thereby forming the adhesive layer. In analternative method, the above dispersion is applied on a peelable sheetand, after drying, the coating is transferred to the support.

When the percutaneous preparation of the present invention is anointment, a cream, or a lotion, examples of the base material include,but not limited to, hydrocarbons such as white petrolatum, liquidparaffin, paraffin, squalane, and plastibase, higher alcohols such ascetanol and stearyl alcohol, higher fatty acids such as isostearic acid,oleic acid, and lauric acid, thickeners such as carboxyvinyl polymer,carboxymethylcellulose, methylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, polyvinyl alcohol, polyacrylic acid,sodium polyacrylate, polyvinyl pyrrolidone, acacia, alginic acid, sodiumalginate, and gelatin, and polyhydric alcohols such as glycerin,propyleneglycol, and 1,3-butyleneglycol.

The percutaneous preparation of the present invention may incorporate,in addition to the components described above, other additives such asanti-oxidants, excipients, solubilizing agents, antibacterial agents,and skin irritation reducing agents in accordance with needs. Examplesof the anti-oxidant include vitamin E and vitamin C. Examples of theexcipients include kaolin, bentonite, and titanium dioxide. Examples ofthe solubilizing agents include α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, and sulfobutylether-β-cyclodextrin. Examples of the antibacterial agents includebenzalkonium chloride, benzoic acid, and methyl parahydroxybenzoate.Examples of the skin irritation reducing agents include silicic acidanhydride. Moreover, absorption regulating agents may be incorporated.Examples of the absorption regulating agents includepolyprenylazacycloalkanes (such as 1-dodecylazacycloheptan-2-one) andfats and oils (such as olive oil, castor oil, jojoba oil, corn germ oil,sunflower oil, coconut oil, squalane, squalene, orange oil, and mineraloil).

EXAMPLES

The present invention will next be described in more detail by way ofReferential Examples, Examples, and Formulation Examples, which shouldnot be construed as limiting the invention thereto.

Referential Example 11-[N-(1S)-1-Ethoxycarbonyl-3-phenylpropyl]-L-alanyl]-L-prolinetert-butyl ester (compound (IV))

Enalapril maleate (5.0 g) was suspended in water (40 mL), and saturatedaqueous sodium bicarbonate solution (60 mL) was added to the suspension,thereby forming a complete solution. The pH of the aqueous solution wasadjusted to 4 to 5 with 10 w/v % hydrochloric acid. The solution wasextracted with chloroform, and the obtained organic layer was dried overanhydrous sodium sulfate. The solvent was removed under reducedpressure, and the residue was dissolved in dioxane. To the solution,concentrated sulfuric acid (1.0 mL) and isobutene (30 mL) were added.The mixture was transferred to a sealable reactor, followed by closingthe reactor, and stirred at room temperature for two days. The reactionmixture was neutralized with saturated aqueous sodium bicarbonatesolution, and the mixture was extracted with ether. The obtained organiclayer was washed with water and saturated brine and dried over anhydroussodium sulfate. The solvent was removed under reduced pressure, and theresidue was purified by silica gel column chromatography (hexane:ethylacetate=1:1), to thereby yield 2.74 g of the title compound as acolorless oily substance.

¹H-NMR (CDCl₃) δ: 1.21-1.38 (6H, m), 1.45 (9H, s), 1.87-2.25 (7H, m),2.59-2.80 (2H, m), 3.24 (1H, t, J=6.6 Hz), 3.43-3.60 (2H, m), 4.18 (2H,q, J=7.2 Hz), 4.41-4.46 (1H, m), 7.14-7.30 (5H, m). IR (neat) ν_(max):2977, 2932, 2875, 1737, 1650, 1453, 1422, 1367, 1154, 1094, 1031, 750,701 cm⁻¹. MS m/z (ESI+): 433 (M+H)⁺.

Referential Example 21-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline tert-butylester (compound (V))

The compound (IV) (2.0 g) was dissolved in methanol (15 mL), and, at 0°C., a 1 mol/L aqueous sodium hydroxide solution (15 mL) was addedthereto, followed by stirring for three hours at room temperature. Thereaction mixture was neutralized with 10 w/v % hydrochloric acid, andmethanol was removed, followed by extraction with chloroform. Theobtained organic layer was dried over anhydrous sodium sulfate. Thesolvent was removed under reduced pressure, to thereby yield 1.87 g ofthe title compound as a colorless amorphous substance.

¹H-NMR (CDCl₃) δ: 1.42 (9H, s), 1.47 (3H, d, J=6.8 Hz), 1.87-2.01 (3H,m), 2.10-2.25 (3H, m), 2.76-2.83 (2H, m), 3.33-3.61 (3H, m), 4.04-4.15(1H, m), 4.36-4.41 (1H, m), 7.10-7.23 (5H, m). IR (KBr) ν_(max): 3435,2981, 1735, 1654, 1450, 1368, 1226, 1152, 1095, 1042, 849, 750, 701cm⁻¹. MS m/z (ESI+): 405 (M+H)⁺. [α]_(D)=−44.2° (CHCl₃, c:1.07).

Referential Example 31-[N-[(1S)-1-Benzyloxycarbonyl-3-phenylpropyl]-L-alanyl]-L-prolinetert-butyl ester (compound (VI))

The compound (V) (1.47 g) was dissolved in N,N-dimethylformamide (7.0mL), and benzyl bromide (684 mg) and potassium carbonate (502 mg) wereadded thereto, followed by stirring for one hour at room temperature.Water (70 mL) was added to the reaction mixture, and the resultantmixture was extracted with ethyl acetate. The obtained organic layer waswashed with water and saturated brine and dried over anhydrous sodiumsulfate. The solvent was removed under reduced pressure, and the residuewas purified by silica gel column chromatography (hexane:ethylacetate=1:1), to thereby yield 1.65 g of the title compound as acolorless oily substance.

¹H-NMR (CDCl₃) δ: 1.25 (3H, d, J=6.8 Hz), 1.43 (9H, s), 1.75-2.25 (7H,m), 3.20-3.52 (4H, m), 4.38 (1H, dd, J=3.6, 8.3 Hz), 5.13 (1H, d, J=12.1Hz), 5.18 (1H, d, J=12.1 Hz), 7.05-7.47 (10H, m). IR (neat) ν_(max):2976, 1736, 1648, 1496, 1454, 1423, 1367, 1154, 750, 700 cm⁻¹.[α]_(D)=−86.4° (CHCl₃, c:1.39)

Referential Example 41-[(N-[(1S)-1-Benzyloxycarbonyl-3-phenylpropyl]-L-alanyl]-L-proline(compound (VII))

The compound (VI) (1.50 g) was dissolved in dichloromethane (6.0 mL),and at 0° C., trifluoroacetic acid (6.0 mL) was added dropwise thereto,followed by stirring for two hours at room temperature. After thesolvent was removed under reduced pressure, the pH of the reactionmixture was shifted to an alkaline range with saturated aqueous sodiumbicarbonate solution, and subsequently adjusted to 4 to 5 with 10 w/v %hydrochloric acid, followed by extraction with chloroform. The obtainedorganic layer was dried over anhydrous sodium sulfate, and the solventwas removed under reduced pressure. The residue was purified by silicagel column chromatography (chloroform:methanol=50:1), to thereby yield1.09 g of the title compound as a colorless amorphous substance.

¹H-NMR (CDCl₃) δ: 1.23 (3H, d, J=6.8 Hz), 1.77-2.33 (6H, m), 2.55-2.78(2H, m), 3.21-3.45 (3H, m), 3.50 (1H, q, J=6.8 Hz), 4.40-4.50 (1H, m),5.11 (1H, d, J=12.1 Hz), 5.17 (1H, d, J=12.1 Hz), 7.06-7.46 (10H, m). IR(KBr) ν_(max): 3448, 3030, 2954, 2879, 1736, 1638, 1497, 1454, 1382,1191, 749, 698 cm⁻¹. [α]_(D)=−86.4° (CHCl₃, c:1.14)

Referential Example 51-[N-[(1S)-1-Benzyloxycarbonyl-3-phenylpropyl]-L-alanyl]-L-proline2-benzyloxyethyl ester (compound (IIa))

The compound (VII) (1.06 g) was dissolved in benzene (5.0 mL), and2-benzyloxyethanol (1.85 g) and p-toluenesulfonic acid monohydrate (555mg) were added thereto, followed by stirring for three hours underreflux while generated water was removed by azeotropy. The solvent wasremoved, and ethyl acetate was added thereto. The mixture was washedwith saturated aqueous sodium bicarbonate solution, water, and saturatedbrine and dried over anhydrous sodium sulfate. The solvent was removedunder reduced pressure, and the residue was purified by silica gelcolumn chromatography (hexane:ethyl acetate=1:1), to thereby yield 1.02g of the title compound as a colorless oily substance.

¹H-NMR (CDCl₃) δ: 1.23 (3H, d, J=6.8 Hz), 1.70-2.35 (7H, m), 2.51-2.77(2H, m), 3.20-3.76 (6H, m), 4.16-4.42 (2H, m), 4.46-4.58 (3H, m), 5.12(1H, d, J=12.1 Hz), 5.17 (1H, d, J=12.1 Hz), 7.04-7.47 (15H, m). IR(neat) ν_(max): 3474, 3325, 3062, 3029, 2953, 2874, 1742, 1645, 1496,1454, 1424, 1366, 1277, 1184, 1028, 916, 746, 700 cm⁻¹. [α]_(D)=−67.6°(CHCl₃, c:1.27)

Referential Example 61-[N-[(1S)-1-Benzyloxycarbonyl-3-phenylpropyl]-L-alanyl]-L-proline3-benzyloxypropyl ester (compound (IIb))

In the same manner as in Referential Example 5, 943 mg of the titlecompound was obtained as a colorless oily substance from 800 mg of thecompound (VII) and 1.52 g of 3-benzyloxypropanol.

¹H-NMR (CDCl₃) δ: 1.23 (3H, d, J=6.8 Hz), 1.75-2.27 (9H, m), 2.54-2.73(2H, m). 3.20-3.59 (6H, m), 4.09-4.29 (2H, m), 4.42-4.54 (1H, m), 4.48(2H, s), 5.12 (1H, d, J=12.1 Hz), 5.17 (1H, d, J=12.1 Hz), 7.04-7.45(15H, m). IR (neat) ν_(max): 3473, 3322, 3061, 3029, 2956, 2871, 1740,1646, 1496, 1454, 1423, 1364, 1277, 1182, 1096, 1046, 1029, 916, 741,699 cm⁻¹. [α]_(D)=−67.3° (CHCl₃, c:1.02)

Referential Example 71-[N-[(1S)-1-Benzyloxycarbonyl-3-phenylpropyl]-L-alanyl]-L-proline4-benzyloxybutyl ester (compound (IIc))

In the same manner as in Referential Example 5, 831 mg of the titlecompound was obtained as a colorless oily substance from 800 mg of thecompound (VII) and 1.64 g of 4-benzyloxybutanol.

¹H-NMR (CDCl₃) δ: 1.24 (3H, d, J=6.6 Hz), 1.56-2.30 (11H, m), 2.55-2.75(2H, m), 3.21-3.59 (6H, m), 4.01-4.20 (2H, m), 4.40-4.54 (1H, m), 4.49(2H, s), 5.12 (1H, d, J=12.1 Hz), 5.18 (1H, d, J=12.1 Hz), 7.03-7.45(15H, m). IR (neat) ν_(max): 3448, 3324, 3061, 3029, 2952, 2869, 1740,1648, 1496, 1454, 1422, 1362, 1277, 1182, 1095, 1055, 1029, 740, 699cm⁻¹. [α]_(D)=−60.9° (CHCl₃, c:1.56)

Referential Example 81-[N-[(1S)-1-Benzyloxycarbonyl-3-phenylpropyl]-L-alanyl]-L-proline2-methoxyethyl ester (compound (IId))

In the same manner as in Referential Example 5, 725 mg of the titlecompound was obtained as a colorless oily substance from 800 mg of thecompound (VII) and 694 mg of 2-methoxyethanol.

¹H-NMR (CDCl₃) δ: 1.25 (3H, d, J=6.8 Hz), 1.79-2.33 (7H, m), 2.54-2.75(2H, m), 3.22-3.73 (6H, m), 3.36 (3H, s), 4.15-4.33 (2H, m), 4.52 (1H,dd, J=3.7, 8.4 Hz), 5.13 (1H, d, J=12.1 Hz), 5.18 (1H, d, J=12.1 Hz),7.07-7.44 (10H, m). IR (neat) ν_(max): 3473, 3322, 3061, 3028, 2952,2880, 1741, 1650, 1604, 1496, 1454, 1423, 1371, 1351, 1278, 1183, 1130,1095, 1032, 751, 700 cm⁻¹. [α]_(D)=−77.8° (CHCl₃, c:1.06)

Referential Example 91-[N-[(1S)-1-Benzyloxycarbonyl-3-phenylpropyl]-L-alanyl]-L-proline2-(2-methoxyethoxy)ethyl ester (compound (IIe))

In the same manner as in Referential Example 5, 675 mg of the titlecompound was obtained as a colorless oily substance from 720 mg of thecompound (VII) and 986 mg of 2-(2-methoxyethoxy)ethanol.

¹H-NMR (CDCl₃) δ: 1.25 (3H, d, J=6.6 Hz), 1.80-2.30 (7H, m), 2.54-2.77(2H, m), 3.29 (2H, t, J=6.6 Hz), 3.37 (3H, s), 3.40-3.76 (8H, m),4.17-4.35 (2H, m), 4.36-4.57 (1H, m), 7.06-7.44 (5H, m). IR (neat)ν_(max): 3481, 3321, 3061, 3028, 2952, 2928, 2878, 1741, 1646, 1454,1423, 1366, 1278, 1184, 1112, 1047, 1030, 972, 917, 850 cm⁻¹.[α]_(D)=−71.1° (CHCl₃, c:1.02)

Referential Example 10 (R)-2-Hydroxy-4-phenylbutyric acid (compound (X))

(R)-2-Hydroxy-4-phenylbutyric acid ethyl ester (compound (IX)) (500 mg)was dissolved in ethanol (5.0 mL), and, at 0° C., a 10 w/v % aqueoussodium hydroxide solution (1.2 mL) was added dropwise thereto, followedby stirring for one hour at 0° C. The solvent was removed, and 1 mol/Lhydrochloric acid was added dropwise to the residue at 0° C., to therebyadjust the pH of the mixture to 3, followed by extraction with diethylether. The obtained organic layer was washed with saturated brine anddried over anhydrous sodium sulfate, and the solvent was removed underreduced pressure, to thereby yield 412 mg of the title compound as acolorless powder.

m.p.: 104-107° C. ¹H-NMR (CD₃OD) δ: 1.85-1.95 (1H, m), 1.97-2.11 (1H,m), 2.74 (2H, t, J=7.9 Hz), 4.08 (1H, dd, J=4.2, 8.3 Hz), 7.13-7.28 (5H,m). IR (KBr) ν_(max): 3459, 2925, 1733, 1242, 1097, 695 cm⁻¹.[α]_(D)=−8.5° (MeOH, c:1.04)

Referential Example 11 (R)-2-Hydroxy-4-phenylbutyric acid benzyl ester(compound (XI))

The compound (X) (1.0 g) was dissolved in N,N-dimethylformamide (10 mL),and potassium bicarbonate (611 mg) and benzyl bromide (660 μL) wereadded thereto at 0° C., followed by stirring for eight hours at 50° C.The solvent was removed, and water was added to the residue, followed byextraction with ethyl acetate. The obtained organic layer was washedwith saturated brine and dried over anhydrous sodium sulfate, and thesolvent was removed under reduced pressure. The obtained crude crystalswere recrystallized from n-hexane, to thereby yield 1.1 g of the titlecompound as colorless crystals.

m.p.: 59-60° C. ¹H-NMR (CDCl₃) δ: 1.89-2.00 (1H, m), 2.07-2.18 (1H, m),2.63-2.81 (2H, m), 4.23 (1H, dd, J=4.0, 7.5 Hz), 5.16 (1H, d, J=12.7Hz), 5.20 (1H, d, J=12.7 Hz), 7.14-7.20 (3H, m), 7.24-7.29 (2H, m),7.35-7.39 (5H, m). IR (KBr) ν_(max): 3458, 2943, 1728, 1450, 1251, 1103,699 cm⁻¹.

Referential Example 12 N-[(1S)-1-(Benzyloxycarbonyl)-3-phenyl]-L-alaninetert-butyl ester maleic acid salt (compound (XII))

The compound (XI) (2.0 g) was dissolved in dichloromethane (20 mL), and2,6-lutidine (0.94 mL) and trifluoromethanesulfonic anhydride (1.4 mL)were added thereto at 0° C., followed by stirring for two hours at 0° C.Chloroform was added to the reaction mixture, and the resultant mixturewas washed with a 5 w/v % aqueous potassium bisulfate solution andsaturated aqueous sodium bicarbonate solution. The obtained organiclayer was washed with saturated brine and dried over anhydrous sodiumsulfate. The solvent was removed under reduced pressure, to therebyyield 3.5 g of 2-trifluoromethanesulfonyloxy-4-phenylbutyric acid benzylester as a brown viscous substance. An L-alanine tert-butyl esterhydrochloride (2.0 g) was dissolved in water (20 mL), and ammoniumcarbonate (1.7 g) and a nitromethane solution (15 mL) of2-trifluoromethanesulfonyloxy-4-phenylbutyric acid benzyl ester (3.5 g)were added to the solution, followed by stirring for three hours at 50°C. Ethyl acetate was added to the reaction mixture, and the resultantmixture was washed with a 5 w/v % aqueous potassium bisulfate solution,saturated aqueous sodium bicarbonate solution, water, and saturatedbrine. The mixture was dried over anhydrous sodium sulfate, and thesolvent was removed under reduced pressure. The residue was dissolved inethyl acetate (14 mL), and maleic acid (859 mg) was added thereto,followed by heating under reflux. After the crystals were completelydissolved, the solution was gradually cooled to room temperature, tothereby yield crude crystals of the title compound. The crude crystalswere recrystallized from ethyl acetate, to thereby yield 2.63 g of thetitle compound as colorless crystals.

m.p.: 137-138° C. ¹H-NMR (CDCl₃) δ: 1.44 (9H, s), 1.48 (3H, d, J=7.2Hz), 2.24 (2H, t, J=7.3 Hz), 2.60-2.79 (2H, m), 3.69 (1H, q, J=7.2 Hz),3.77 (1H, t, J=6.2 Hz), 5.20 (1H, d, J=11.9 Hz), 5.27 (1H, d, J=11.9Hz), 6.34 (1H, s), 7.08-7.10 (2H, m), 7.18-7.28 (3H, m), 7.38-7.39 (5H,m). IR (KBr) ν_(max): 2987, 1746, 1458, 1352, 1162, 996, 697 cm⁻¹.[α]_(D)=−1.43° (CHCl₃, c:1.38)

Referential Example 13 N-[(1S)-1-(Benzyloxycarbonyl)-3-phenyl]-L-alanine(compound (XIII))

Water was added to compound (XII) (2.5 g), and the pH of the mixture wasadjusted to 8 with saturated aqueous sodium bicarbonate solution,followed by extraction with ethyl acetate. The obtained organic layerwas washed with saturated brine and dried over anhydrous sodium sulfate.The solvent was removed under reduced pressure, and a hydrogenchloride-dioxane solution (45 mL) was added dropwise thereto, followedby stirring for 13 hours at room temperature. The solvent was removedunder reduced pressure, water was added thereto, and the pH of themixture was adjusted to 6 with saturated aqueous sodium bicarbonatesolution. The formed precipitate was collected through filtration andthen recrystallized from ethyl acetate, to thereby yield 1.5 g of thetitle compound as a colorless powder.

m.p. : 160-161° C. ¹H-NMR (CD₃OD) δ: 1.48 (3H, d, J=7.2 Hz), 2.14-2.21(2H, m), 2.52-2.62 (1H, m), 2.67-2.77 (1H, m), 3.57 (1H, q, J=7.2 Hz),4.05 (1H, t, J=6.1 Hz), 5.18 (1H, d, J=11.9 Hz), 5.36 (1H, d, J=11.9Hz), 7.08-7.27 (5H, m), 7.36-7.45 (5H, m). IR (KBr) ν_(max): 2781, 1739,1618, 1356, 1262, 1192, 698 cm⁻¹ [α]_(D)=−6.6° (MeOH, c:1.10)

Referential Example 14 L-Proline 3-benzyloxypropyl ester oxalate(compound (XIV))

L-Proline (2.30 g) was dissolved in benzene (20 mL), andp-toluenesulfonic acid monohydrate (4.57 g) and 3-benzyloxypropanol(1.58 mL) was added thereto, followed by stirring for 15 hours underreflux while generated water was removed by azeotropy. The solvent wasremoved, and saturated aqueous sodium bicarbonate solution was addedthereto, followed by extraction with chloroform. The extract was washedwith saturated brine and dried over anhydrous sodium sulfate. Thesolvent was removed under reduced pressure, and the residue wasdissolved in ethyl acetate (16 mL). Oxalic acid (693 mg) was dissolvedin the solution under heating, and the resultant solution was left tostand at room temperature. The formed crystals were collected throughfiltration and then recrystallized from ethyl acetate, to thereby yield1.92 g of the title compound as a colorless powder.

m.p. : 84-85° C. ¹H-NMR (CDCl₃) δ: 1.90-2.11 (5H, m), 2.13-2.45 (1H, m),3.41-3.50 (1H, m), 3.52 (2H, t, J=6.0 Hz), 4.24-4.39 (2H, m), 4.43-4.51(1H, m), 4.48 (2H, s), 7.30-7.38 (5H, m). IR (KBr) ν_(max): 3348, 2873,1742, 1637, 1560, 1458, 1402, 1279, 1229, 1108, 720, 497 cm⁻¹[α]_(D)=−28.7° (CHCl₃, c:1.17)

Referential Example 151-[N-[(1S)-1-Benzyloxycarbonyl-3-phenylpropyl]-L-alanyl]-L-proline3-benzyloxypropyl ester (compound (IIb))

The compound (XIV) (500 mg) was dissolved in water (6 mL), and the pH ofthe solution was adjusted to 8 with saturated aqueous sodium bicarbonatesolution, followed by extraction with chloroform. The obtained organiclayer was washed with saturated brine and dried over anhydrous sodiumsulfate. The solvent was removed under reduced pressure, and the residuewas dissolved in chloroform (7 mL). At 0° C., the compound (XIII) (483mg), N,N′-dicyclohexylcarbodiimide (291 mg), and1-hydroxy-1H-benzotriazole monohydrate (237 mg) were added to thesolution. The temperature of the solution was gradually increased from0° C. to room temperature, and the solution was stirred for 23 hours atroom temperature. The reaction mixture was washed with a 5 w/v % aqueouspotassium hydrogensulfate solution, saturated aqueous sodium bicarbonatesolution, water, and saturated brine, and subsequently dried overanhydrous sodium sulfate. The solvent was removed, and the residue waspurified by silica gel column chromatography (hexane:ethyl acetate=2:3to 1:2), to thereby yield 747 mg of the title compound as a colorlessoily substance.

Example 1 1-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline2-hydroxyethyl ester (Inventive Compound 1)

The compound (IIa) (2.65 g) was dissolved in ethanol (100 mL), 5 mass %palladium carbon (1.3 g) was added to the solution, followed by stirringfor 13 hours at room temperature under hydrogen atmosphere (4 atm). Thecatalyst was removed by filtration with Celite, and the filtrate wascondensed under reduced pressure. The residue was purified throughsilica gel column chromatography (chloroform:methanol=3:1), to therebyyield 1.49 g of the title compound as a colorless amorphous substance.

¹H-NMR (CDCl₃) δ: 1.47 (3H, d, J=6.8 Hz), 1.80-2.36 (6H, m), 2.60-2.83(2H, m), 3.27-3.95 (6H, m), 3.96-4.57 (4H, m), 6.97-7.22 (5H, m). IR(KBr) ν_(max): 3422, 3028, 2956, 2879, 1742, 1655, 1560, 1543, 1509,1498, 1451, 1388, 1282, 1187, 1087, 1051, 895, 861, 753, 702 cm⁻¹. MSm/z (ESI+): 393 (M+H)⁺. [α]_(D)=−26.4° (CHCl₃, c:1.20)

Example 2 1-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline3-hydroxypropyl ester (Inventive Compound 2)

In the same manner as in Example 1, 2.06 g of the title compound wasobtained as a colorless amorphous substance from 2.97 g of the compound(IIb).

¹H-NMR (CDCl₃) δ: 1.55 (3H, d, J=7.0 Hz), 1.78-2.38 (8H, m), 2.65-2.87(2H, m), 3.31 (1H, t, J=7.0 Hz), 3.36-3.80 (5H, m), 4.00-4.15 (1H, m),4.17-4.39 (2H, m), 4.47-4.60 (1H, m), 7.07-7.28 (5H, m). IR (KBr)ν_(max): 3385, 3027, 2958, 2878, 1741, 1655, 1560, 1543, 1509, 1498,1439, 1388, 1280, 1186, 1093, 1053, 920, 863, 751, 702 cm⁻¹. MS m/z(ESI+): 407 (M+H)⁺. [α]_(D)=−33.0° (CHCl₃, c:1.04)

Example 3 1-[N-[(1S)-1-Carboxy-3-phenylpropyl)-L-alanyl]-L-proline4-hydroxybutyl ester (Inventive Compound 3)

In the same manner as in Example 1, 2.92 g of the title compound wasobtained as a colorless amorphous substance from 4.51 g of the compound(IIc).

¹H-NMR (CDCl₃) δ: 1.35-2.31 (10H, m), 1.49 (3H, d, J=6.8 Hz), 2.63-2.83(2H, m), 3.33 (1H, t, J=6.8 Hz), 3.38-3.77 (5H, m), 3.88-4.55 (4H, m),7.00-7.24 (5H, m). IR (KBr) ν_(max): 3386, 3028, 2953, 2874, 1742, 1656,1451, 1382, 1281, 1214, 1184, 1093, 1046, 944, 859, 752, 701 cm⁻¹. MSm/z (ESI+): 421 (M+H)⁺. [α]_(D)=−32.9° (CHCl₃, c:1.31).

Example 4 1-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline2-methoxyethyl ester (Inventive Compound 4)

The compound (IId) (700 mg) was dissolved in ethanol (4.0 mL), and 5mass % palladium carbon (140 mg) was added to the solution, followed bystirring for one hour at room temperature under hydrogen atmosphere(normal pressure). The catalyst was removed through filtration withCelite, and the filtrate was condensed under reduced pressure. Theresidue was purified by silica gel column chromatography(chloroform:methanol=20:1), to thereby yield 547 mg of the titlecompound as a colorless amorphous substance.

¹H-NMR (CDCl₃) δ: 1.45 (3H, d, J=7.0 Hz), 1.86-2.35 (6H, m), 2.64-2.86(2H, m), 3.25 (1H, t, J=7.0 Hz), 3.36 (3H, s), 3.42-3.69 (4H, m), 3.82(1H, q, J=6.8 Hz), 4.15-4.43 (2H, m), 4.54 (1H, dd, J=3.5, 8.3 Hz),7.06-7.25 (5H, m). IR (KBr) ν_(max): 3448, 3028, 2954, 2882, 1743, 1655,1560, 1543, 1523, 1509, 1498, 1450, 1380, 1280, 1185, 1129, 1094, 1034,916, 865, 753, 702 cm⁻¹. MS m/z (ESI+): 407 (M+H)⁺. [α]_(D)=−44.5°(CHCl₃, c:1.10)

Example 5 1-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline2-(2-methoxyethoxy)ethyl ester (Inventive Compound 5)

In the same manner as in Example 4, 465 mg of the title compound wasobtained as a colorless amorphous substance from 640 mg of the compound(IIe).

¹H-NMR (CDCl₃) δ: 1.43 (3H, d, J=6.8 Hz), 1.87-2.32 (6H, m), 2.64-2.85(2H, m), 3.22 (1H, t, J=6.4 Hz), 3.37 (3H, s), 3.43-3.80 (8H, m),4.17-4.45 (2H, m), 4.55 (1H, dd, J=3.5, 8.3 Hz), 7.09-7.28 (5H, m). IR(KBr) ν_(max): 3448, 3027, 2930, 2880, 1742, 1655, 1560, 1543, 1523,1509, 1498, 1451, 1382, 1281, 1187, 1139, 1109, 1048, 919, 859, 752, 703cm⁻¹. MS m/z (ESI+): 451 (M+H)⁺. [α]_(D)=−41.1° (CHCl₃, c:1.05)

Example 6

Stability Test

About 5 mg of each of Inventive Compounds 1 to 5 and enalapril was putin a glass vial. Isopropyl myristate (100 μL) was added, to therebyyield a solution (or suspension). The vial was tightly sealed and storedin a 60° C. thermostatic chamber for one week. Methanol was added to thesolution contained in the vial until the total volume of the solutionmixture became 100 mL. The resultant solution was employed as a testsolution to be subjected to an HPLC analysis. Peak area ratio (expressedby percentage) of each compound with respect to a sum of all theobtained peak areas was calculated. The calculation show that, assummarized in Table 1, enalapril had been completely degraded into itscorresponding closed-ring product or other degraded products over thestorage term of one week, whereas the Inventive Compounds exhibited goodstability.

TABLE 1 Area ratio (%): storage at 60° C. for 1 week Proline ClosedOther ester ring degraded Compound compound Enalapril Enalaprilatproduct products Inventive 1 91.8 — 0.0 0.0 8.2 Compound 2 96.7 — 0.00.0 3.3 3 94.4 — 0.0 0.0 5.6 4 99.1 — 0.0 0.0 0.9 5 94.2 — 0.0 0.0 5.8Comp. Example Enalapril — 0.0 0.0 48.4 51.6

Example 7

Metabolism Test by Use of Cultured Human Skin

A sample of a three-dimensional cultured human skin model (LSE-high,Toyobo Co., Ltd.) was set in a vertical diffusion cell whose temperaturewas maintained at 37° C., and the receptor phase of the cell was filledwith 7 mL isotonic phosphate buffer (PBS, pH 7.4). The cell was allowedto stand for one hour. Each of Inventive Compounds 1 to 3 and enalapril(serving as a Comparative Example) was dissolved or suspended inisopropyl myristate so as to attain a concentration of 1.0 mass %. Thesolution (200 μL) was applied to the skin sample, whereby the teststarted. When 24 hours had passed after the test started, theconcentrations of proline esters or enalapril (i.e., a prodrug ofenalaprilat) which remained unchanged, and the transformed productenalaprilat contained in PBS in the receptor phase were determinedthrough HPLC. From the obtained data, concentration ratio of “prolineesters or enalapril”: “enalaprilat” was calculated (presence ratio). Asshown in Table 2, of the proline esters which permeated thethree-dimensional cultured human skin model, 64 to 77% was converted toenalaprilat.

TABLE 2 Presence ratio^(a)) Inventive Compound 1 23.5/76.5 InventiveCompound 2 36.3/63.7 Inventive Compound 3 30.4/69.6 Enalapril (Comp.Ex.) 98.8/1.2 ^(a))Concentration ratio (proline ester orenalapril/enalaprilat)

Example 8

Production of Patches

Styrene-isoprene-styrene block copolymer (trade name; Quintac 3421,product of Zeon Corporation) (30 g) and a tackifying resin (an aliphaticsaturated hydrocarbon resin, trade name: Quintone M100, product of ZeonCorporation) (60 g) were dissolved in toluene (110 g), and liquidparaffin (10 g) was added thereto, followed by further mixing so as toform a uniform solution, to thereby prepare an adhesive solution. Theadhesive solution (65 mass %) was mixed with Inventive Compound 1 (20mass %), isopropyl myristate (10 mass %), and lauromacrogol (5 mass %),the “mass %” being based on dried solid content of the formed coating.The viscosity of the mixture was adjusted with toluene, followed bymixing so as to form a uniform solution. The drug-containing adhesivesolution was applied through use of a film applicator to thesilicone-coated side of a peelable polyethylene terephthalate sheet(thickness 75 μm) so that the formed coating has a thickness of 200 μm.The product was dried for ten minutes at approximately 65° C., and apolyethylene terephthalate support (thickness 12 μm) was affixed to thecoating surface. The product was cut into pieces having predetermineddimensions, to thereby prepare patch 1.

Similarly, Inventive Compounds 2 to 5 were processed, thereby preparingpatches 2 to 5, respectively.

Hairless Mouse Skin Permeability Test

A portion of the skin was extirpated from of each male hairless mouse (5weeks old, body weight: about 20 g) and placed in a vertical diffusioncell maintained at 37° C. PBS (pH 7.2, 7 mL) was charged to the receptorphase, and the skin sample was allowed to stand for one hour. Each ofthe patches 1 to 5 was punched out, to thereby provide patch pieces(1.33 cm²). Each patch piece was attached to each skin sample, wherebythe test was started. PBS (0.5 mL) from the receptor phase was sampledhourly, from the start of the test to hour 8. Thereafter, the samplingwas performed every two hours to hour 24. After each sampling wascomplete, PBS was replenished to the receptacle in the same amount asthe sampled PBS. Each of the PBS sample collected from the receptaclewas analyzed through HPLC so as to determine the concentrations ofunchanged proline esters (i.e., prodrugs of enalaprilat) and transformedproduct enalaprilat. The determined proline ester concentration wasregarded as an enalaprilat concentration, and total enalaprilatconcentration was calculated through addition of two enalaprilatconcentrations. The total concentration was plotted with respect topermeation time. From the obtained permeation profile, skin permeationrate (flux) was determined from the slope of the curve in a region wherepermeation rate was in a steady state. In addition, permeation delaytime (Lag time) and 24-hour-cumulative permeation amount per unit patcharea (1 cm²) were also calculated. The concentration (in PBS in thereceptacle) ratio of proline ester: enalaprilat 24 hours after the startof the test was determined, thereby serving as presence ratio of prolineester: enalaprilat. As shown in Table 3, the percutaneous preparationcontaining a proline ester of the present invention exhibited excellentskin permeability, and the proline ester permeated the skin wasconverted to enalaprilat in a proportion of about 50 to 85%.

TABLE 3 Skin Cumulative Tape Permeation Permeation permeation form ratedelay time Presence amount drug (μg/cm²/hr) (hr) ratio^(a)) (μg/cm²)Patch 1 21.80 3.60 50.5/49.5 393.39 Patch 2 25.03 2.59 29.3/70.7 443.64Patch 3 17.30 2.40 41.9/58.1 374.31 Patch 4 6.14 4.20 20.2/79.8 88.88Patch 5 11.59 2.30 15.0/85.0 187.10 ^(a))Concentration ratio (prolineester/enalaprilat)

1. A proline ester represented by the following formula (I):

wherein R¹ represents a hydroxy-lower alkyl group, a lower alkoxy-loweralkyl group, or a lower alkoxy-lower alkoxy-lower alkyl group or apharmaceutically acceptable salt thereof.
 2. The proline ester asdescribed in claim 1, which is selected from the group consisting of1-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline 2-hydroxyethylester, 1-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline3-hydroxypropyl ester,1-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline 4-hydroxybutylester, 1-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline2-(2-methoxyethoxy)ethyl ester, and1-[N-[(1S)-1-Carboxy-3-phenylpropyl]-L-alanyl]-L-proline 2-methoxyethylester, or a pharmaceutically acceptable salt thereof.
 3. A drugcomprising a proline ester as recited in claim 1 or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier.
 4. Apercutaneous preparation comprising a proline ester as recited in claim1 or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 5. The percutaneous preparation as described inclaim 4, which is a patch.
 6. The percutaneous preparation as describedin claim 4, which comprises one or more percutaneous absorptionenhancers selected from the group consisting of a fatty acid ester and anon-ionic surfactant.
 7. The percutaneous preparation as described inclaim 6, wherein the percutaneous absorption enhancer is selected fromthe group consisting of isopropyl myristate, lauromacrogol, lauric aciddiethanolamide, glyceryl monocaprylate, glyceryl monolaurate, sorbitanmonocaprylate, and polyoxyethylene sorbitan monooleate.
 8. A method fortreating a pathological condition affected or induced by activation ofan ACE comprising: administering to a subject in need thereof aneffective amount of a proline ester of claim 1 or a pharmaceuticallyacceptable salt thereof; wherein the pathological condition is selectedfrom the group consisting of hypertension, a cardiac disease selectedfrom the group consisting of cardiac hypertrophy, cardiac failure, andmyocardial infarct.
 9. The method of claim 8, wherein administration isperformed percutaneously.
 10. The proline ester of claim 1, wherein R¹represents a hydroxy-lower alkyl group.
 11. The proline ester of claim1, wherein R¹ represents a lower alkoxy-lower alkyl group.
 12. Theproline ester of claim 1, wherein R¹ represents a lower alkoxy-loweralkoxy-lower alkyl group.